Characterization of a spontaneous mouse retinal pigment epithelial cell line B6-RPE07.

PURPOSE. A spontaneously arising retinal pigment epithelial (RPE) cell line (B6-RPE07) was cloned from a primary culture of mouse RPE cells and maintained in culture for more than 18 months. Morphologic and functional properties of this cell line have been characterized.

METHODS. The morphology of the B6-RPE07 cells was examined by phase-contrast light microscopy, electron microscopy, and confocal microscopy. Barrier properties were measured by the flux of fluorescence from the apical to the basolateral compartment of culture chambers. The abilities of the cells to bind/phagocytose photoreceptor outer segments (POS) were determined by confocal microscopy, electron microscopy, and flow cytometry. Cytokine/chemokine secretion was measured by cytometric bead array. The expression of visual cycle proteins was determined by RT-PCR and Western blotting.

RESULTS. In standard culture conditions, B6-RPE07 cells display cobblestone morphology. When cultured on three-dimensional (3D) collagen gel–coated membranes, B6-RPE07 cells exhibit a monolayer epithelial polarization with apical surface microvilli. Immunohistochemistry of B6-RPE07 cultures revealed a high expression of pan-cytokeratin. B6-RPE07 cells also expressed the retinal pigment epithelium-specific marker CRALBP, but not RPE65. Cell junction proteins ZO-1 and ß-catenin, but not claudin-1/3 or occludin-1, were observed in B6-RPE07 cells. B6-RPE07 cells are able to bind, phagocytose, and digest POS. Finally, B6-RPE07 cells produce high levels of IL-6 and CCL2.

CONCLUSIONS. This is the first report of a mouse RPE cell line with morphology, phenotype, and function similar to those of in vivo mouse RPE cells. This cell line will be a valuable resource for future RPE studies, in particular for in vivo gene modification and transplantation studies.

Decision making by senior social workers at point of first referral

In the United Kingdom there has been difficulty in implementing the family support provisions contained in the 1989 Children Act, largely because of continued emphasis on child protection activity by local authorities. There is an observable international tendency for child-care referrals to receive investigative response, resulting in families being traumatized and children's needs left unmet. There has been a lack of research into how child-care referrals are initially categorized by senior social workers. This paper reports on research undertaken in two Health and Social Services Trusts within Northern Ireland to ascertain if it might be possible to treat more initial referrals as 'child-care problem enquiries' as opposed to 'child protection investigations'. Results demonstrate that, while such potential may exist, a preoccupation with the management of risk could lead to the development of child-care problems receiving quasi-child protection responses. Consequently, changes in initial decision making may not have the full intended effects in terms of the organizational release of resources for family support or a lessening of the traumatic impact upon families.

Retinal Endothelial Cell Apoptosis Stimulates Recruitment of Endothelial Progenitor Cells

PURPOSE. Bone marrow–derived endothelial progenitor cells (EPCs) contribute to vascular repair although it is uncertain how local endothelial cell apoptosis influences their reparative function. This study was conducted to determine how the presence of apoptotic bodies at sites of endothelial damage may influence participation of EPCs in retinal microvascular repair.

METHODS. Microlesions of apoptotic cell death were created in monolayers of retinal microvascular endothelial cells (RMECs) by using the photodynamic drug verteporfin. The adhesion of early-EPCs to these lesions was studied before detachment of the apoptotic cells or after their removal from the wound site. Apoptotic bodies were fed to normal RMECs and mRNA levels for adhesion molecules were analyzed.

RESULTS. Endothelial lesions where apoptotic bodies were left attached at the wound site showed a fivefold enhancement in EPC recruitment (P < 0.05) compared with lesions where the apoptotic cells had been removed. In intact RMEC monolayers exposed to apoptotic bodies, expression of ICAM, VCAM, and E-selectin was upregulated by 5- to 15-fold (P < 0.05–0.001). EPCs showed a characteristic chemotactic response (P < 0.05) to conditioned medium obtained from apoptotic bodies, whereas analysis of the medium showed significantly increased levels of VEGF, IL-8, IL-6, and TNF-a when compared to control medium; SDF-1 remained unchanged.

CONCLUSIONS. The data indicate that apoptotic bodies derived from retinal capillary endothelium mediate release of proangiogenic cytokines and chemokines and induce adhesion molecule expression in a manner that facilitates EPC recruitment.

Appearance of the frequency-doubling stimulus at threshold.

PURPOSE. It has been argued that the threshold for detecting frequency-doubling (FD) technology perimeter stimuli differs from the threshold for perceiving spatial structure (pattern) in the same targets. Thresholds for perceiving spatial structure have typically been assessed using orientation-identification experiments. The authors investigated the influence of orientation, edge profile, and psychophysical method on the origin of the reported differences in detection and orientation-identification thresholds for FD gratings.

METHODS. Detection and orientation-identification thresholds were determined in 12 observers with the use of FD stimuli (0.25 cyc/deg, 25 Hz) presented centrally and at 15° eccentricity. Edge profile (square- and Gaussian-windowed) and orientation (horizontal, vertical, and oblique) were independently modified. Detection thresholds were also measured for spatially uniform flickering targets (25 Hz). Orientation-identification thresholds using a two-alternative forced choice (2-AFC) and a two-interval forced choice (2-IFC) method were also compared in five experienced observers.

RESULTS. Orientation-identification and detection thresholds did not significantly differ under any condition tested. Orientation-identification thresholds obtained with 2-AFC were not significantly different from those obtained with 2-IFC. Thresholds for spatially uniform flicker were significantly lower than for FD stimuli.

CONCLUSIONS. The authors found that orientation-identification and detection thresholds for FD gratings did not differ and argue that recent findings to the contrary arise from the inappropriate use of spatially uniform flicker targets as alternatives in 2-IFC experiments.

Immune activation in Retinal Aging: A Gene Expression Study.

PURPOSE
To investigate changes in gene expression during aging of the retina in the mouse.

METHODS
Total RNA was extracted from the neuroretina of young (3-month-old) and old (20-month-old) mice and processed for microarray analysis. Age-related, differentially expressed genes were assessed by the empiric Bayes shrinkagemoderated t-statistics method. Statistical significance was based on dual criteria of a ratio of change in gene expression >2 and a P < 0.01. Differential expression in 11 selected genes was further verified by real-time PCR. Functional pathways involved in retinal ageing were analyzed by an online software package (DAVID-2008) in differentially expressed gene lists. Age-related changes in differential expression in the identified retinal molecular pathways were further confirmed by immunohistochemical staining of retinal flat mounts and retinal cryosections.

RESULTS
With ageing of the retina, 298 genes were upregulated and 137 genes were downregulated. Functional annotation showed that genes linked to immune responses (Ir genes) and to tissue stress/injury responses (TS/I genes) were most likely to be modified by ageing. The Ir genes affected included those regulating leukocyte activation, chemotaxis, endocytosis, complement activation, phagocytosis, and myeloid cell differentiation, most of which were upregulated, with only a few downregulated. Increased microglial and complement activation in the aging retina was further confirmed by confocal microscopy of retinal tissues. The most strongly upregulated gene was the calcitonin receptor (Calcr; >40-fold in old versus young mice).

CONCLUSIONS
The results suggest that retinal ageing is accompanied by activation of gene sets, which are involved in local inflammatory responses. A modified form of low-grade chronic inflammation (para-inflammation) characterizes these aging changes and involves mainly the innate immune system. The marked upregulation of Calcr in ageing mice most likely reflects this chronic inflammatory/stress response, since calcitonin is a known systemic biomarker of inflammation/sepsis. © Association for Research in Vision and Ophthalmology.